Mouse model for aging

ABSTRACT

A mouse model for mammalian aging is disclosed. In one embodiment, the invention comprises a mouse having a genomic mutation in the exonuclease domain II (ExoII) of a mitochondrial DNA polymerase gamma (POlG) gene, wherein the mutation leads to high levels of mutations in polymerase mtDNA.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. provisional Ser. No.60/663,930, Mouse Model for Aging, Tomas Prolla, et al., filed Mar. 21,2005, incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States government support awarded byNIH R01 AG021905. The United States government has certain rights inthis invention.

BACKGROUND OF THE INVENTION

Mitochondria are intracellular organelles that are the main source ofATP, and play a central role in several metabolic processes includingfatty acid β-oxidation, phospholipid biosynthesis, calcium signaling andreactive oxygen species (ROS) generation. Mitochondria also play acritical role in the process of apoptosis, since numerous pro-apoptoticmolecules and pathological stimuli converge on these organelles leadingto outer membrane permeabilization and cell death (D. R. Green and G.Kroemer, Science 305:626-9, 2004).

Because mitochondria are the main source of ROS in the cell and theorganelle contains its own ˜16 kbp circular DNA, a central role formtDNA mutations in aging has been postulated (J. E. Fleming, et al.,Gerontology (Basel) 28:44-53, 1982). Indeed, mtDNA has a higher mutationrate as compared to nuclear DNA (D. C. Wallace et al., Curr. Genet.12:81-90,1987) and mtDNA mutations have been shown to accumulate withaging in several tissues of various species (Y. Wang, et al., Proc.Natl. Acad. Sci. USA 98:4022-4027, 2001; S. Melov, etal., Nucleic AcidsRes. 25:974-982, 1997; M. Corral-Debrinksi, et al., Nat.Genet.2:324-329, 1992; C. M. Lee, et al., J. Gerontol. Biol. Sci.48:B201-B205, 1993; M. Khaidakov, et al., Mutat. Res. 526:1-7, 2003),and in cells derived from aged humans (Y. Michikawa, et al., Science286:774-779, 1999).

In agreement with a central role for mitochondria in aging, a systematicRNA interference (RNAi) screen of C. elegans for gene inactivations thatincrease lifespan has revealed genes important for mitochondrialfunction (S. S. Lee, et al., Nat. Genet. 33:40-48, 2003), and inhibitionof mitochondrial activity in this organism affects lifespan (A. Dillin,et al., Science 298:2398-2401, 2002).

BRIEF SUMMARY OF THE INVENTION

In the present application, we construct mice engineered to have a highmtDNA mutation rate and provide support for a causal role of apoptosisin aging due to mtDNA mutations. We also provide an improved mouse modelfor aging.

In one embodiment, the present invention is a transgenic mouse model formammalian aging comprising a mouse having a genomic mutation in anexonuclease domain, preferably the exonuclease domain II (ExoII), of amitochondrial DNA polymerase gamma (Polg) gene (SEQ ID NO: 1), whereinthe mutation is capable of maintaining mitochondrial DNA polymeraseactivity and impairing the exonuclease domain activity. Preferably, themutation is selected from the group consisting of a single or doublebase pair nucleotide substitution.

In another embodiment, the transgenic mouse has a genomic mutation in anexonuclease domain, preferably the exonuclease domain II (ExoII), of amitochondrial DNA polymerase gamma (Polg) gene, wherein the mutation isa nucleotide change within residues 819-836,1026-1067 or 1413-1442 ofthe mouse Polg gene (as numbered in NM_(—)017462 or SEQ ID NO: 1), andwherein the mutation is capable of maintaining mitochondrial DNApolymerase replication activity and impairing the exonuclease domainactivity.

Preferably, the mutation is a double base substitution resulting in achange in the coding of residue 257 from an aspartic acid (D) to analanine (A) in the exonuclease domain II of Polg.

In a preferred version of the invention, the mouse model exhibitssymptoms of accelerated or premature aging compared to a mouse nothaving the mutation in the exonuclease domain of the Polg gene. Theaging symptoms are preferably selected from the group consisting ofabnormalities in tissues of high cellular turnover, heart dysfunction,graying hair and alopecia, auditory function loss, cochleardegeneration, immune cell loss, anemia, male germ cell loss leading tolack of sperm and infertility, skeletal muscle mass loss (sarcopenia),neurodegeneration, increased presence of apoptotic markers, and loss ofbone mass.

In another embodiment, the present invention. is a method of screeningfor a potentially therapeutic agent useful for delaying the onset ofaging-related symptoms. The method typically comprises the steps of: (a)providing a mouse model as described above, wherein the mouse exhibitsaging-related symptoms; (b) administering the agent to the mouse model;and (c) determining whether the agent is capable of delaying the onsetof aging-related symptoms in the mouse model treated with the agentcompared to an untreated mouse model.

In another embodiment, the present invention is a method of screeningfor a potentially therapeutic agent useful for treating medicalconditions comprising progressive external ophthalmoplegia, sensorimotorpolyneuropathy, ataxia, Parkinson's syndrome or early menopause definedby mitochondrial DNA mutations in a POLG gene. The method typicallycomprises the steps of: (a) providing a mouse model as described above,wherein the mouse exhibits symptoms of progressive externalophthalmoplegia sensorimotor polyneuropathy, ataxia, Parkinson'ssyndrome or early menopause; (b) administering the agent to the mousemodel; and (c) determining whether the agent is capable of improvingsymptoms for any of the medical conditions of step (a) in the mousemodel treated with the agent compared to an untreated mouse model.

In another embodiment, the present invention is a method for generatinga mouse model for mammalian aging, typically comprising the steps of:(a) introducing into a mouse a germline transmission of a mutation in anexonuclease domain, preferably exonuclease domain II of a mitochondrialDNA polymerase gamma (Polg) gene, wherein the mutation results in adecrease in the Polg exonuclease activity, and wherein the mutationresults in the production of a PolgA^(exo−/+)mouse; (b) crossbreedingthe PolgA^(exo−/+)mouse of step (a); and (c) generating a homozygousPolgA^(exo−/+)mouse which exhibits at least one symptom of aging,wherein the symptoms of aging are selected from the group consisting ofabnormalities in tissues of high cellular turnover, heart dysfunction,graying hair and alopecia, auditory function loss, cochleardegeneration, immune cell loss, anemia, male germ cell loss leading tolack of sperm and infertility, skeletal muscle mass loss (sarcopenia),loss of bone mass, neurodegeneration, increased presence of apopticstress markers, and decreased mitochondrial function.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1. D257A mice display a premature aging phenotype. Shown arewild-type (A) and D257A mice (B and C) at ˜13 months of age. Progeroidsymptoms including hair loss, graying, and kyphosis become apparent at˜9 months of age and progress rapidly thereafter. (D) Kaplan-Meiersurvival curve of cohorts of wild-type (+/+), D257A heterozygous mice(D257A/+) and D257A homozygous mice (D257A/D257A). Mice were of a mixed129/ICR/B6 genetic background. At least 230 mice per genotype arerepresented in the survival curves.

FIG. 2. D257A mice develop abnormalities in tissues of high cellularturnover. Shown are H&E-stained sections from wild-type (A and C) andD257A (B and D) duodenum and cecum at ˜10 months of age. Large regionsof the cecum display reduced number of crypts (B) and aberrant vilarfusion and branching is observed in the duodenum (D). Testicular crosssections of wild-type (E) and D257A (F) showing loss of spermatogoniaand sperm in D257A animals at 10 months of age. Abnormal testiculararchitecture is observed initially at ˜5 months of age. The thymus isreduced in size (G) and weight (H) in D257A mice at 3 months of age(*P<0.0003).

FIG. 3. mtDNA mutations and replicative senescence. Mouse embryonicfibroblasts (MEF) were derived from individual embryos. Cultures werepassaged using a 1:4 subculture regimen and incubated at 37° C. in anormoxic atmosphere of 75% N₂, 5% CO₂, and 20% O₂ (A and B) or a hypoxicatmosphere of 93% N₂, 5% CO₂, and 2% O₂ (C and D). Wild-type cell lines:22, 23, 35, 48, 49 and 50. D257A cell lines: 25, 36 and 38.

FIG. 4. Age-related loss of auditorv function and cochlear degenerationin D257A mice. Cochlea from wild-type (A, B and C) and D257A (D, E andF) animals. Arrows in panel A indicate hair cell and spiral ganglionneuronal cell regions shown in panels B and C respectively. D257A micedisplay preservation of cochlear hair cells (E) and severe loss ofspiral ganglion neuronal cells (F) by 9 months of age. (G and H) Meansof auditory-evoked brain stem response (ABR) thresholds (dB SPL) forwild-type and D257A mice at 4, 8 and 16 kHz. Means are not differentbetween 2-month-old wild-type and D257A mice (G), but are markedlyelevated in 9-month-old D257A mice (N=5, P<0.0001) (H), indicating lossof auditory function.

FIG. 5. Age-related heart dysfunction in D257A mice. Echocardiograms of9-10 month-old wild-type (A) and D257A mice (B). Anterior and posteriorwalls are relative to instrument location (chest). Wave forms representindividual heart beats, with maximal and minimal chamber size at systole(s) and diastole (d) respectively indicated by arrows. Aging in D257Amice is associated with heart chamber enlargement and loss ofcontractile function, as determined by impaired diastole (d). (C) LVDd,left ventricular diameter at diastole. FRS, % fractional shortening.IVRT, isovolumic relaxation time. Nine wild-type and D257A mice wereused for measurements. *P<0.05.

FIG. 6. Oxidative stress markers in isolated mitochondria and tissuesfrom D257A mice. (A) hydrogen peroxide production in isolatedmitochondria from wild-type and D257A mice at ˜9 months of age. (B)Protein carbonyl levels, a marker of protein oxidation, was measured bywestern blotting in isolated mitochondria from wild-type and D257A miceat 9 months of age. (C) F2-isoprostanes, a marker of lipid peroxidation,was measured by gas chromatography/negative ion chemical ionization massspectrometry in liver and skeletal muscle (vastus lateralis) tissuesfrom 6-month-old wild-type and D257A mice. (D) Oxidative damage to DNA(8-oxo-dG) and RNA (8-oxo-G), was measured by HPLC in liver tissue of9-month-old wild-type and D257A mice. Nine wild-type and D257A mice wereused for hydrogen peroxide production, mitochondrial carbonyl analysis,and nucleic acid oxidative damage assays. Six wild-type and D57A micewere used for F2-isoprostanes measurements. *P<0.05.

FIG. 7. Caspase activation. Activation of caspase-3 requires proteolyticprocessing of its inactive zymogen into activated fragments. Thespecific antibody used detects endogenous levels of the large fragment(17/19 kDa) of activated caspase-3 resulting from cleavage adjacent toAsp175. Extracts of tissues (25 μg) from wild-type and D257A animals ofthe indicated ages were separated by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and probed with a mouse monoclonal antibodyagainst cleaved caspase 3. Units are arbitrary OD units. *For alltissues tested, the difference between wild-type and D257A mice wassignificant (P<0.05).

FIG. 8. Apoptosis detection by TUNEL. Arrows indicate TUNEL-positiveapoptotic nuclei. Quantification of apoptosis in thymus (A), smallintestine (B) and testis (C) of wild-type (left panels) and D257A(center panels). The number of apoptotic nuclei per 10⁵ μm² (thymus),100 villi (intestine) and tubule cross section (testis) were counted inH&E-stained sections from the indicated genotypes. Each bar representsapoptotic nuclei from intestinal, thymus and testis sections of at least4 mice/genotype. *P<0.05.

FIG. 9. Generation of D257A mice. (A) A ˜12.3 Kb genomic regioncontaining all three exonuclease domain encoding sequences of POLG,ExoI, ExoII and ExoIII is shown. We constructed a targeting vectorcontaining two arms of homologous DNA, approximately 5 Kb in lengtheach. The neo selectable marker was flanked with loxp sites (which arenot drawn to scale). On the 5′ targeting vector arm we introduced an ACto CT double base substitution at positions 1054-55 of the mouse PolgAmRNA using a PCR based site-directed mutagenesis strategy, resulting inthe introduction of a novel Xhol site. (B) Structure of targeted PolgAlocus following successful homologous recombination. (C) Mice carryingthe the D257A mutation were crossed to Cre recombinase expressingtransgenic mice, resulting in the removal of the region flanked by IoxPsites, including the neo selectable marker. Only a small regioncontaining a single IoxP site remains after recombination. (D) Southernanalysis of gene targeting in ES cells. Following electroporation, EScells clones were selected in G418 containing media and expanded. DNAextracted from individual clones was digested with Nsil and Clal, andprobed with a 5′ external probe. The asterisk denotes an ES clone thatcarries both the targeted polgA^(D257Aneo) and wild-type alleles.

FIG. 10. Body weight and bone mineral density as a function of age. (A)Body weight of males, (B) Body weight of females, (C) Bone mineraldensity (femur) in males and females combined. Wild-type and D257A miceat approximately 6 months and 10 months of age were anesthetized with240 mg/kg tribromoethanol, secured to an lightly adhesive tray andsubjected to X-ray densitometery using a PIXImus™ densitometer (GELunar, Madison Wis.). Bone mineral density (BMD) was calculated usingPIXImus software version 1.45.

FIG. 11. Sequencing of mtDNA in various tissues of wild-tvpe and D257Amice. We isolated mitochondria from heart, duodenum (duo) and liver fromfrom individual wild-type and D257A animals at 5-6 months of age.Mitochondrial DNA was isolated and digested with DraIII and BgIIIfollowed by treatment with exonuclease III in order to degrade anycontamination with nuclear DNA. A 525 bp mtDNA fragment that spans aportion of the D-loop region and mt-Cytb (cytochrome b) gene was PCRamplified and cloned into the pCR4-TOPO cloning vector. One hundredninety-two clones from each tissue source, representing ˜100,000 bp,were sequenced and the DNA sequences aligned using Aligner software(Codon Code). The frequency of PCR- and cloning-induced mutations (1/16,000 bp), was determined by reamplifying, cloning and sequencingindividual clones. (A) Tissue distribution of mtDNA mutations inwild-type and D257A mice after background substraction. Two animals wereused for each tissue/genotype. (B) Mutational spectrum in wild-type andD257A mice. Transition mutations represented the largest class ofmutations in all tissues examined. Transversions varied greatly intissues of wild-type mice (3-26%), and represented a higher proportionof mutations in the heart (26%) of wild-type tissues. The proportion oftransversions was higher (16-25%) and more uniform in the D257A tissues,consistent with the fact that most mtDNA mutations in D257A mice arelikely to arise from the same mechanism, nucleotide misincorporation byPOLG. The total number of mutations represented in the graph ranged from110 (sperm, wild-type) to 637 (heart, D257A).

FIG. 12. Red blood cell counts. (A) Red blood cell counts in individualwild-type and D257A mice plotted versus age. (B) Mean red blood cellcounts in young and old wild-type mice. Means were compared by t test;*P<0.05.

FIG. 13. Organ Weights. (A) Organ weights in wild-type and D257A mice at3 months of age. (B) Organ weights in wild-type and D257A mice at 3months of age. Means were compared by t test; *P<0.05.

DETAILED DESCRIPTION OF THE INVENTION

In General

Mutations in mitochondrial DNA (mtDNA) accumulate in a variety oftissues of mammalian species and contribute to aging through poorlydefined mechanisms. We have addressed this issue by characterizing micethat express a proof-reading deficient version of the mitochondrial DNApolymerase gamma (Polg) and accumulate mtDNA mutations in at least twotissues, preferably in both mitotic and post-mitotic tissues describedbelow. Accelerated aging in these animals is characterized by markedalterations in organs displaying rapid cellular turnover, such as theintestinal epithelium, testis and thymus, as well as loss of skeletalmuscle mass, heart dysfunction, hearing loss, graying and alopecia.Accumulation of mtDNA mutations is not associated with increased markersof oxidative stress, but is correlated with the induction of apoptosisin target tissues.

We have engineered an animal model of accelerated aging. The presentinvention broadly relates to a transgenic mouse model for mammalianaging including a mouse having a genomic mutation in an exonucleasedomain of a mitochondrial DNA polymerase gamma (Polg) gene. We envisionthat mutations in the ExoI, ExoII or ExoIII domains would be suitable,as described below.

Mouse Model

Specifically, one embodiment the present invention provides for atransgenic mouse model for mammalian aging. Preferably, the mouse has agenomic mutation in an exonuclease domain, preferably ExoII, of amitochondrial DNA polymerase gamma (Polg) gene. The impairment of theexonuclease domain activity results from the expression of a DNAproof-reading deficient version of the mitochondrial DNA polymerasegamma (Polg) gene and accumulation of mitochondrial DNA mutations inboth mitotic and post-mitotic mouse tissues, which is correlated withthe activation of caspase-3 and the induction of apoptosis in mousetissues.

The examples below show creation of a POLG mutation in mice derived from129 SV AB2.2ES cells. However, we believe that the mutation can beintroduced into any mouse strain with successful results because thesame type of mutation would work similarly in any mouse strain. As withthe mouse model described below in the Examples, one would typicallytarget one of the exonuclease domains of mitochondrial DNA polymerasegamma (Polg) gene, preferably that gene described at NM_(—)017462. N.Lecrenier, et al., Gene 185:147-152, 1997, incorporated by reference,discloses the sequence and characterization of mitochondrial DNApolymerase.

By “exonuclease domain” we mean nucleotides at 819-836, 1026-1067 or1413-1442, as numbered in NM_(—)017462 or SEQ ID NO:1. SEQ ID NO:2 isthe amino acid sequence of the wild-type POL gene. SEQ ID NO:3 is thesequence of a preferred POLG mutations, D257A.

The examples below show the creation of the genetically manipulated“knock-in” mouse from embryonic stem cells. This is a preferred methodof creating a transgenic mouse of the present invention.

In one preferred embodiment, the mutation is a double base substitution(AC to CT) at residues 1054 to 1055 of ExoII in the mouse Polg gene, asnumbered in NM_(—)017462. However, we believe that other mutations willbe equally suitable for the present invention. As described above, themouse must accumulate mtDNA mutations in at least two tissues. One mayreview the examples to determine how this accumulation is best measured.The present invention specifically includes other mutations that willresult in a mt mutation accumulation. For example, one may wish to dosingle or double base mutations between residues 819-836 (ExoI),1026-1067 (ExoII) or 1413-1442 (ExoIII) of the POLG gene.

The mouse model exhibits symptoms of aging which include, but are notlimited to, abnormalities in tissues of high cellular turnover, heartdysfunction, graying hair and alopecia, auditory function loss, cochleardegeneration, immune cell loss, anemia, male germ cell loss leading tolack of sperm and infertility, skeletal muscle mass loss (sarcopenia),neurodegeneration, increased presence of oxidative stress markers, anddecreased mitochondrial function. The primary symptoms would be thefollowing: impaired hearing function and heart function, loss of boneand muscle mass and, at the molecular basis, the induction of apoptosis.

Method of Screening

Another embodiment of the invention provides a method of screening for apotentially therapeutic agent useful for delaying the onset ofaging-related symptoms. The method includes providing a mouse model asdescribed above, wherein the mouse exhibits aging-related symptoms andadministering the agent to the mouse model. One would then determinewhether the agent is capable of delaying the onset of aging-relatedsymptoms in the mouse model treated with the agent compared to anuntreated mouse model. Specifically, one may wish to examine eithersingly or a set of symptoms selected from the group consisting ofabnormalities in tissues of high cellular turnover, heart dysfunction,graying hair and alopecia, auditory function loss, cochleardegeneration, immune cell loss, anemia, male germ cell loss leading tolack of sperm and infertility, skeletal muscle mass loss (sarcopenia),neurodegeneration, loss of bone mass and increased presence of oxidativestress markers. Preferably, one should administer the compound to themice early in life, and measure survival rates and organ function laterin life.

In a related embodiment, the invention provides a method of screeningfor a potentially therapeutic agent useful for treating medicalconditions such as progressive external ophthalmoplegia, sensorimotorpolyneuropathy, ataxia, Parkinson's syndrome or early menopause. Theagent could be an agent that acted at the genetic level, at apharmaceutical level or is a dietary product.

Generation of a Mouse Model

In yet another embodiment, the invention provides a method of generatinga mouse model for mammalian aging. The method includes introducing intoa mouse a germlne transmission of a mutation in an exonuclease domain,preferably exonuclease domain 11, of a mitochondrial DNA polymerasegamma (Polg) gene as described above. Preferably, the mutation is adouble base substitution (AC to CT) at residues 1054 to 1055 of themouse Polg gene. The mutation results in the production of apolg^(exo−/+)mouse. One would then typically crossbreed thepolg^(exo−/exo +)mouse and generate a homozygous polg^(exo−/exo−)mousewhich exhibits at least one symptom of aging, wherein the symptoms ofaging are selected from the group consisting of abnormalities in tissuesof high cellular turnover, heart dysfunction, graying hair and alopecia,auditory function loss, cochlear degeneration, immune cell loss, anemia,male germ cell loss leading to lack of sperm and infertility, skeletalmuscle mass loss (sarcopenia), neurodegeneration, increased presence ofoxidative stress markers, and decreased mitochondrial function.

EXAMPLES Mitochondrial DNA Mutations, Oxidative Stress and Apoptosis inMammalian Aging

mtDNA mutations lead to a premature aging phenotvpe. Mitochondrial DNAreplication in mammalian cells lacks DNA mismatch repair, relying solelyon the 3′-5′ exonuclease activity of POLG to correct DNA mismatches thatarise due to nucleotide misincorporation during DNA replication. Wecloned the locus encoding the catalytic subunit of mouse POLG, PolgA,and used gene targeting in embryonic stem cells to introduce a AC to CTtwo base substitution at position 1054-1055 of the PolgA cDNA (FIG. 9).This mutation results in a critical aspartate to alanine residuesubstitution in the second conserved exonuclease domain of POLG. Thissubstitution profoundly impairs the proof-reading ability of mouse POLG(A. Trifunovic, et al., supra, 2004), and the equivalent mutation inyeast POLG results in a marked increase in the mtDNA mutation rate (F.Foury and S. Vanderstraeten, EMBO Journal 11:2717-26,1992). Germlinetransmission of the mutation resulted in the production ofPolgA^(D257A/+) mice, which were intercrossed to generate homozygousPolgA^(D257A/D257A) mice, hereafter denoted D257A. Although we expectedthat D257A mice would display a mtDNA mutator phenotype, we observed noevidence of embryonic lethality or developmental abnormalities in theseanimals. Young D257A mice were indistinguishable from wild-typelittermates, but long-term follow up revealed a striking premature agingappearance beginning at ˜9 months of age, consisting of hair loss,graying and kyphosis (FIG. 1A to C). These observations were associatedwith a reduced lifespan (maximum survival of 460 days as comparedwith >852 days for wild-type littermates, P<0.0001, FIG. 1D). We alsoobserved an age-related decrease in body weight and bone density (FIG.S2), which is a hallmark of aging in rodents (H. R. Massie, et al., Exp.Gerontol. 25:469-481, 1990) and humans (E. Seeman, J. Appl. Physiol.95:2142-2151, 2003). Although initially fecund, we observed sterility ofboth male and female D257A animals at ˜5 months of age.

To examine if mtDNA mutations accumulate to an equal extent in mitoticand post-mitotic tissues of D257A mice, we performed large-scalesequencing of a 525 bp region of mtDNA that spans the control region anda fragment of the gene encoding cytochrome b. Tissues were derived fromheart, liver, duodenum and sperm of ˜6-month-old D257A mice andwild-type controls. DNA sequencing of over 300,000 bp of mtDNA for eachtissue revealed that D257A mice display a frequency of mtDNA mutationsin this region that is ˜3 times the level observed in wild-type animalsfor all tissues examined except sperm, which displayed only a slightincrease (˜35%) in mutation frequency (FIG. 11). Possibly, selection forviable sperm results in the elimination of sperm that carry high mtDNAmutational loads. Most mutations were transition mutations, with thelevel of transversions varying greatly in wild-type tissues (3-26%), andless so in D257A tissues (16-25%) (FIG. 11). These observations suggestthat different mechanisms contribute to the mutational spectrum of mtDNAin various tissues of wild-type mice, but reduction of mtDNA replicationfidelity is the major mutational mechanism in mtDNA of D257A mice.Surprisingly, we find that the frequency of mtDNA mutations in6-month-old wild-type mice is as high as 6.7×10⁻⁴ mutations/bp,representing over 10 mutations/mitochondrial genome. Our findingsindicates that large levels of mtDNA mutations exist in adult animals inboth mitotic and post-mitotic tissues, and that further age-relatedaccumulation of mtDNA mutations may contribute to several agingphenotypes. The modest increase (˜3-fold) in mtDNA mutation frequencyreported here for D257A mice is surprising in view of the dramaticphenotype observed in these animals, the severe impact of this mutationon exonuclease activity in vitro (A. Trifunovic, et al., supra, 2004),and the marked increase in mitochondrial mutation frequency in yeastcarrying the equivalent mutation (F. Foury, S. Vanderstraeten, supra,1992). This observation suggests that mitochondria that harbor largenumbers of mutated mtDNA molecules are degraded, or that cells thatcarry large numbers of such mutations do not survive.

mtDNA mutations impact tissues of high cellular turnover. We nextperformed necropsy and histological examination of D257A mice of variousages. A remarkable phenotype was dysplasia of the intestinal epithelium,characterized by an age-related loss of crypts in some regions of thececum (FIG. 2A and B), villar fusion, and complex villar branching inregions of the duodenum (FIG. 2C and D). The intestinal epithelium is atissue of very high cellular turnover, and it appears to be particularlysusceptible to the accumulation of mtDNA mutations and subsequent lossof tissue homeostasis. This finding is of interest given the recentreport of accumulation of pathogenic mtDNA mutations in human coloniccrypt stem cells with aging (R. W. Taylor, et al., J. Clin. Invest.112:1351-1360, 2003). We also observed reduced testicular size and aprogressive depletion of spermatogonia in the testis of D257A mice,leading to a degeneration of the seminiferous epithelium by ˜6 months ofage (FIG. 2E and F). Sperm are generated continuously throughoutreproductive life through germlne cell division, and therefore germlnestem cells are likely to accumulate high levels of mtDNA mutations inD257A mice.

Thymic atrophy is a common aging phenotype in several mammalian species.We observed that the earliest detectable phenotype of D257A mice is aprofound thymic atrophy starting at 2-3 months of age (FIG. 2G). Thymusweight is 40.6±4.4 mg in wild-type mice as compared to 16.0±1.88 mg inD257A mice at 3 months of age (P<0.0003, FIG. 2H). Hematopoietic stemcell precursors of mature T-cells originate from the bone marrow andmigrate to the thymus, which is the major site of T-lymphocytematuration. Within the cortex of the thymus, precursors of T cellsundergo extensive cell proliferation, but more than 95% of thymocytesdie by apoptosis before they reach the medulla and the blood stream (K.Abbas, A. H. Litchman, J. S. Pober, in Cellular and MolecularImmunology, A. K. Abbas, A. H. Litchaman, J. S. Pober, Eds. (Saunders,Philadelphia, 1997), pp. 171-193). Thus, thymic atrophy in D257A may bedue to reduced cellular proliferation, increased apoptosis, or both. Wedid not observe overt depletion of hematopoietic cells in the bonemarrow of D257A mice. However, loss of bone marrow homeostasis seemslikely given that red blood cell counts were reduced significantly inD257A mice as a function of age (FIG. 12). Hair graying in aging inhumans and mice has recently been linked to loss of melanocyte stemcells (E. K. Nishimura, et al., Science 2004 Dec 23; Epub ahead ofprint), and D257A mice display early onset graying (FIG. 1 B and C).When taken as a whole, this collection of phenotypes suggests thataccelerated aging associated with mtDNA mutations in D257A mice is atleast partly due to depletion of stem cell progenitors and loss ofhomeostasis in highly proliferative tissues.

One possible mechanism for the D257A phenotypes described above is adefect in cellular proliferation associated with the accumulation ofmtDNA mutations. In order to characterize the effects of the D257Amutation and subsequent accumulation of mtDNA mutations on replicativesenescence, we derived several independent mouse embryonic fibroblastslines (MEFs) from D257A and wild-type littermates and measured thenumber of cell doublings prior to senescence. Passaging of wild-typeMEFs under normal, high-oxygen tension (20%) leads to rapid senescenceassociated with oxygen toxicity, whereas passaging MEFs under low oxygentension (2%) does not lead to replicative senescence (S. Parrinello, etal., Nat. Cell Biol. 5:741-747, 2003). At 20% oxygen, both wild-type andD257A MEFs underwent rapid senescence (FIG. 3A and B). In contrast, wedid not observe senescence or reduced growth of either wild-type orD257A MEFs following over 40 days of tissue culture (FIG. 3C and D).Thus, accelerated aging in D257A mice is not likely to be due to anintrinsic defect in cellular proliferation.

mtDNA mutations result in accelerated age-related hearing loss,sarcopenia and heart dysfunction. We next examined post-mitotic tissuesknown to play critical roles in mammalian aging. Age-related hearingloss (presbycusis) is a hallmark of aging in multiple species, includingmice (Q. Y. Zheng, et al., Hear. Res. 130:94-107,1999), and in humans itaffects over 60% of individuals aged 70 or older (M. A. Gratton and A.E. Vazquez, Curr. Opin. Otolaryngol. Head Neck Surg 11:367-371, 2003).Presbycusis is associated with the age-related accumulation of mtDNAmutations in auditory tissue, although a causal nature for thisrelationship has not been established (M. D. Seidman, et al., Arch.Otolaryngol. Head Neck Surg 123:1039-1045,1997; M. D. Seidman,Laryngoscope 110:727-738, 2000). Hearing loss can be monitored by anelevation in auditory-evoked brainstem responses (ABR). We conducted anABR threshold analysis in young (2 months) and old (9 months) D257A miceand wild-type littermates. Our results revealed no difference inauditory function between wild-type and D257A mice at 2 months of age(FIG. 4G), but we found marked elevation of ABR thresholds at 4, 8 and16 kHz (P<0.0001) in D257A mice by 9 months of age, indicating severehearing loss (FIG. 4H). Histological analysis revealed age-related lossof spiral ganglion neurons (FIG. 4C and F), a common feature ofage-related hearing loss (S. L. McFadden, et al., Audiology 40:313-321,2001; E. M. Keithley, et al., Hear. Res. 188:21-28, 2004). Thus, theage-related accumulation of mtDNA mutations can have a causal role inpresbycusis.

Aging in rodents (J. Wanagat, etal., FASEB J. 15:322-332, 2001), rhesusmonkeys (N. G. Gokey, et al., Aging Cell 3:319-326, 2004) and humans (J.Lexell, et al., J. Neurol. Sci. 84:275-294,1988) is also characterizedby loss of muscle mass (sarcopenia) and associated frailty. This loss ofmuscle mass has been correlated with the accumulation of mtDNA mutations(J. W. Pak, et al., Aging Cell 2:1-7, 2003), but there is no directevidence linking age-related, sporadic mtDNA mutations and sarcopenia.Consistent with a causal role for mtDNA mutations in sarcopenia, weobserved muscle loss in 9-month-old D257A mice (FIG. 13). Specifically,muscle weights were significantly reduced in both gastrocnemius(p<0.002, -10% decrease) and quadriceps (p<0.005, -10% decrease) musclesof D257A mice at 9 months of age (FIG. S5). We note that this differencewas not observed between D257A and wild-type mice at 3 months of age(FIG. 13), and also that at 9 months of age D257A mice are not cachexic,indicating that the decline in muscle mass is age-related and not likelyto be an indirect consequence of physiological decline in D257A mice.Therefore, age-related accumulation of mtDNA mutations is likely to be acontributing factor in age-related sarcopenia and associated frailty.

The heart is one of the most energy demanding organs in mammals, with upto 35% of cardiomyocyte volume taken up by the mitochondria (R. P.Crisman, et al., Am. J. Physiol. 248:H8-H14, 1985). Because of thisdependence of the heart on high mitochondrial density, one might expectimpaired structure and/or function in the heart of D257A animals. Infact, mtDNA mutations accumulate with aging in the heart, but to whatextent this contributes to either age-associated cardiac pathology orthe decline in heart function that occurs with normal aging is notclear. Previously reported histological data in D257A mice (Trifunovic,et al., supra, 2004) and mice carrying a heart-specific mutator POLG (D.Zhang, et al., Genomics 69:151-161, 2000) suggests that mtDNA mutationslead to age-related heart hypertrophy. To assess left ventricular (LV)systolic and diastolic function as well as structure, we used in vivoM-mode and Doppler echocardiography in D257A and wild-type controls at12 months of age. We observed increased heart chamber size and LVdilation in D257A mice (FIG. 5A and B). Additionally, we noted that thisdilation was associated with normal wall thickness but a significantdecrease in percent fractional shortening (FRS), a measure of heartcontraction (FIG. 5B). Thus, D257A mice have a dilated, hypocontractileLV hypertrophy. Interestingly, isovolumic relaxation time (IVRT), ameasure of diastolic function, was significantly prolonged in D257Aanimals (FIG. 5B), consistent with the impaired LV relaxation thatoccurs with normal aging in rodents and humans (D. A. Brenner, et al.,Circulation 104:221-226, 2001; P. Spirito and B. J. Maron, Br. Heart J.59:672-679, 1988; P. S. Hees, et al., Am. J. Physiol. Heart Circ.Physiol. 286:H782-J788, 2004).

mtDNA mutations and oxidative stress. It is estimated that 2 to 3% ofthe oxygen consumed by aerobic cells results in the production ofsuperoxide (O₂−), which is converted to hydrogen peroxide (H₂O₂) bysuperoxide dismutase. The basic tenet of the free radical theory ofaging (D. Harman, J. Gerontol. 11:298-300, 1956) is that aging-relatedloss of function is due to the progressive accrual of damage inflictedby ROS. Importantly, mitochondria are thought to produce most cellularROS, and ROS can induce damage to DNA, lipids and proteins. The originalhypothesis has been refined with a ROS mtDNA mutation hypothesis,according to which aging may be caused by accumulation of ROS-mediatedmtDNA mutations over time (J. E. Fleming, et al., supra, 1982). It isfurther postulated that accumulation of mtDNA mutations leads to a“vicious cycle”, further increasing ROS generation and mitochondrialdysfunction (J. E. Fleming, et al., supra, 1982; M. K. Shigenaga, etal., Proc. Natl. Acad. Sci. USA 91:10771-10778,1994). We tested thishypothesis by isolating mitochondria from the heart and liver of youngand old (3 months vs. 9 months) D257A mice and wild-type littermates andmeasuring hydrogen peroxide production. Surprisingly, we found that H₂O₂production was decreased in heart mitochondria of 9-month-old D257A mice(FIG. 6A). We also assayed mitochondrial protein carbonyls, a marker ofoxidative damage to proteins, and found no significant differences inD257A and wild-type animals in mitochondrial (FIG. 6B) or cytosolicfractions of either heart and liver (data not shown). Thus, isolatedmitochondria from D257A animals do not show signs of increased oxidativestress.

Because reduced mitochondrial function and associated energetic deficitsmay reduce cellular antioxidant capacity, we also examined markers ofROS production in tissue extracts of D257A mice. Lipid peroxidation is acentral mechanism of ROS-mediated cellular injury, and can be assessedthrough the measurement of F2-isoprostanes, prostaglandin-like compoundsformed in vivo from the free radical-initiated peroxidation ofarachidonic acid (L. J. Roberts, 2^(nd) and J. D. Morrow, Cell. Mol.Life Sci. 59:808-820, 2002). We used a highly sensitive gaschromatography/mass-spectrometry method to measure F2-isoprostanes inliver and skeletal muscle, and observed no differences between D257Amice and wild-type controls (FIG. 6C). We next examined oxidative damageto DNA and RNA in 9-month old wild-type and D257A mice through themeasurement of 8-hydroxyguanosine and 8-hydroxydeoxyguanosine usingHPLC. We observed no difference in the levels of 8-hydroxydeoxyguanosinelevels in liver DNA (FIG. 6D), and observed a significant reduction inthe steady state levels of 8-hydroxyguanosine in liver RNA of D257A mice(FIG. 6D, P<0.05). Thus, our observations do not support the hypothesisthat accumulation of mtDNA mutations contributes to increased ROSproduction in aging. Instead, our findings demonstrate that agingmediated by mtDNA mutations can occur in the absence of increasedoxidative damage to DNA, protein or lipids.

Apoptosis underlies aging induced by mtDNA mutations. The centralsignaling component of apoptosis is a proteolytic system involving afamily of cysteine proteases called caspases. In the mitochondrialpathway of apoptosis, mitochondrial dysfunction can lead tomitochondrial outer membrane permeabilization (MOMP), the release ofcytochrome c into the cytosol, and activation of caspase-3, a keyeffector caspase found activated in cells committed to death (D. W.Nicholson, et al., Nature 376:3743,1995). To determine the extent ofapoptosis in tissues of D257A mice, we monitored the levels of cleavedcaspase-3 in cytosolic fractions. Levels of cleaved caspase-3 weresignificantly elevated in the cytosolic fractions of testis, duodenum,and thymus of 3-month-old D257A mice (FIG. 7), and this inductionpreceded overt degeneration in these tissues. In skeletal muscle, levelsof cleaved caspase 3 were not significantly altered in 3-month-old D257Amice, but were significantly increased at 9 months of age (FIG. 7), whenanimals displayed loss of muscle mass. Thus, we hypothesize thatactivation of caspase-3 and subsequent apoptosis is likely to be acausal event in loss of tissue homeostasis due to age-relatedaccumulation of mtDNA mutations in multiple tissues of D257A mice.

Apoptosis is also associated with nuclear DNA fragmentation andformation of mono- and oligo-nucleosomes. Because the intestinalepithelium, thymus and testis were severely affected in D257A mice, wealso examined these tissues with terminal transferase-mediated dUTPnick-end labeling (TUNEL), an assay that detects apoptotic cells insitu. Counting of TUNEL positive cells in the duodenum, testis andthymus of 3-month old D257A mice revealed a consistent trend forelevation of TUNEL positive cells in all tissues examined, and thistrend was associated with higher variability in individual D257A mice,as expected from the stochastic nature of mtDNA mutations (FIG. 8). Wealso determined by ELISA that the content of cytosolic mono- andoligo-nucleosomes (180-base pair nucleotides or multiples) wassignificantly increased in testis and thymus of 3-month-old D257A mice(data not shown). Taken as a whole, our observations strongly suggestthat loss of critical, irreplaceable cells through apoptosis is thecentral mechanism of tissue dysfunction associated with the accumulationof mtDNA mutations.

Conclusion. The finding that a two-base substitution in the PolgA genein the context of a 3×10⁹ bp mammalian genome results in theacceleration of multiple age-related phenotypes is striking. Ourobservations in D257A mice and those recently described by Trifunovic,et al. in a mouse model that carries the same mutation (A. Trifunovic,et al., supra, 2004) provide strong evidence for a role of mtDNAmutations in organismal aging. Our findings demonstrate that the D257Amutation leads to accumulation of high levels of mtDNA mutations intissues of high cell turnover, such as the intestine, resulting in lossof tissue homeostasis. We have also functionally characterizedpost-mitotic tissues in D257A mice, and uncovered defects in heart andauditory function, two systems highly affected by aging in humans. Thewide tissue distribution of phenotypes in D257A mice suggests that theaccumulation of mtDNA mutations associated with aging is likely to exertserious functional consequences, and that mtDNA mutations contribute toage-related physiological decline. Importantly, we have demonstratedthat accelerated aging through a mitochondrial pathway can occur in theabsence of increased ROS production, and may be causally linked toincreased apoptosis.

The concept that DNA damage contributes to aging is supported by thefinding that humans and mice carrying mutations in several genesinvolved in DNA repair, including XPD (J. de Boer, et al., Science296:1276-1279, 2002), Ku86 (H. Vogel, et al., Proc. Natl. Acad. Sci. USA96:10770-10775, 1999), and WRN (S. Chang, et al., Nat. Genet.36:877-882, 2004), display progeroid syndromes. It is likely thatseveral types of DNA damage contribute to the aging process (P. Hastyand J. Vijg, Aging Cell 3:55-65, 2004), and our findings suggest thatincreased apoptosis may be a central mechanism for accelerated aging.The finding that increased mutations in mtDNA result in aging phenotypesin D257A mice can be readily correlated with normal aging, since mtDNAmutations clearly accumulate to high levels during aging (Y. Wang, etal., supra, 2001; S. Melov, et al., supra, 1997; M. Corral-Debrinksi, etal., supra, 1992; C. M. Lee, et al., supra, 1993; M. Khaidakov, et al.,supra, 2003). Surprisingly, our findings do not support one of the majortenets of the free radical hypothesis of aging because accumulation ofmtDNA mutations in D257A animals does not lead to increased oxidativestress in either isolated mitochondria or tissues. Because of thestochastic nature of mtDNA mutation accumulation, it is possible thatthere is a wide distribution of mtDNA mutational load in cells of anygiven tissue, and that the cells most affected do produce increased ROSlevels. However, the observed decrease in hydrogen peroxide productionin isolated mitochondria from the heart of D257A mice suggests thatreduced mitochondrial function associated with increased mtDNA mutationsleads to an overall reduction in mitochondrial ROS production. Possibly,this observation reflects adaptive alterations in mitochondria in thecontext of an increasing mtDNA mutational load.

If mtDNA mutations contribute to age-related tissue dysfunction, whatmitochondrial adaptations contributed to the evolution of long-livedanimals? Because cells contain a mixture of wild-type and mutant mtDNAs(heteroplasmy), mtDNA mutations are likely to need to reach a criticalthreshold prior to cellular dysfunction and apoptosis. It is likely thatlong-lived organisms evolved mechanisms to minimize the mtDNA mutationalload and its consequences because the rate of accumulation ofmitochondrial mutations is much faster in mice as compared to humans (E.Wang, et al., Mutat. Res. 377:157-166, 1997). Because POLG is a highlyconserved enzyme, it appears unlikely that differences in the fidelityof mtDNA replication account for the different rates of accumulation ofmtDNA mutations in various organisms. Possibly, the mtDNA mutationalload and its consequences are regulated at multiple levels, includingmtDNA repair pathways, ROS production and the elimination ofdysfunctional mitochondria. Adaptations that increase the mitochondrialapoptotic threshold, such as increased ROS detoxification or modulationof the p53 and p66shc (F. Orsini, et al., J. Biol. Chem.279:25689-25695, 2004) mediated mitochondrial apoptotic pathways, mayalso have an impact on lifespan in mammals in part by suppressing theeffects of mtDNA mutations. In humans, several genetic disordersassociated with mitochondrial mutations have been described which areoften associated with severe age-related functional decline in tissueswith high metabolic demands, including skeletal muscle and brain (D. C.Wallace, Science 283:1482-1488,1999). Mutations of POLG in humans leadto a complex multisystem disorder that can be associated withprogressive ophtalmoplegia, sensorimotor polyneuropathy, ataxia,parkinsonism and early menopause (P. Luoma, et al., Lancet 364, 875-882,2004; M. Filosto, et al., Arch. Neurol. 60:1279-1284, 2003; M. Mancuso,et al., Neurology 62:316-318, 2004). The most severe POLG mutations aredominant and result in alterations in the DNA polymerase domain,resulting in reduced DNA polymerase activity and a 2- to 35-folddecrease in nucleotide selectivity in vitro (M. A. Graziewicz, et al.,Nat. Struct. Mol. Biol. 11:770-776, 2004). Mutations in the exonucleasedomain of POLG have also been reported in humans (M. Filosto, et al.,supra, 2003), and these are associated with reduced DNA replicationfidelity in vitro and accumulation of point mutations in vivo (R. DelBo, et al., Neurology 61:903-908, 2003). Therefore, similar to ourobservations in D257A mice, it is likely that accumulation of sporadicmtDNA mutations in humans contributes to the physiological declineassociated with aging.

A definitive evaluation of the impact of mtDNA mutations to normal agingin specific species will require the generation of organisms that areengineered to have reduced mtDNA mutation rates (G. M. Martin and L. A.Loeb, Nature 429:357-359, 2004). Based on our observations, wehypothesize that mtDNA mutations result in increased apoptosis, leadingto functional decline in postmitotic tissues and depletion of stem cellrenewal capacity in tissues that display rapid cell turnover. Inagreement with this hypothesis, caloric restriction, the onlynutritional intervention that retards aging, retards the accumulation ofmtDNA mutations in primates and rodents (E. Bua, et al., FASEB J.18:582-584, 2004; L. E. Aspnes, et al., FASEB J. 11:573-581, 1997), andreduces mitochondria-mediated apoptotic pathways (H. Y. Cohen, et al.,Science 305:390-392, 2004). The availability of mitochondrial mutatormice should allow for the identification of genetic, nutritional andpharmacological interventions that reduce the impact of mtDNA mutationsin mammalian aging.

Materials and Methods

Generation of D257A mice.

We used a human cDNA fragment (kindly provided by Dr. W. Copeland)encoding the conserved exonuclease domain region of POLG to screen aλFIXII mouse 129Sv genomic library (Stratagene). We identified twopositive clones that hybridized to the cDNA. DNA restriction enzymemapping was performed on both clones, which demonstrated that theycorrespond to overlapping regions in the mouse genome. One clone, λGK-1was used for further characterization. We sequenced the entire 12.3 Kbinsert and found that it encoded exons 1 through 18 (FIG. 9). Thisparticular genomic fragment contained sequences encoding all threeconserved exonuclease domains of POLG: ExoI, ExoII and ExoIII. Weconstructed a targeting vector containing two arms of homologous DNA,approximately 5 Kb each. On the 5′ targeting vector arm we introduced anAC to CT double base substitution at positions 1054-55 of the mousePolgA mRNA using a PCR based site-directed mutagenesis strategy. Theentire fragment used in this strategy was sequenced to confirm thatthere were no additional base substitutions introduced by the PCRstrategy. The substitution changes the coding of residue 257 fromaspartic acid (D) to alanine (A), and introduces in the process a Xholrestriction enzyme site at this position. As a selectable marker forgene targeting, we used a phosphoglycerate kinase promoter-drivenneomycin phosphotransferase (neo) gene. Insertion of the neo cassette isdesigned to occur within intronic DNA (see FIG. 9). We named this allelePolgA^(D257Aneo). We also flanked this neo cassette with Cre recombinaserecognition (IoxP) sites, which were used for Cre recombinase-mediatedexcision of the flanked neo cassette in vivo (see below). We named theexcised allele PolgA^(D257A).

Once constructed, the targeting vector was linearized and introducedinto 129Sv AB2.2 ES cells (a gift from Dr. A. Bradley). Four hundred twoneomycin-resistant ES cell clones were expanded and frozen into 96-wellplates. Southern analysis (Fig. S1) of these clones using a 3′ probeidentified a targeting frequency of ˜11% (43 positives out of 402 clonesscreened). The Southern assay involves detection of an 11.5 Kb targetedallele, as compared to a 15.6 Kb wild-type fragment in a Clal/Nsildouble digest of genomic DNA (FIG. 9). We next performed a PCR assayacross the relevant exon 3 region to determine which targeted clones hadacquired the desired AC to CT double base substitution at positions1054-55. PCR with the chosen primers results in an 857 bp product whichcontains no Xhol sites in wild-type PolgA, but which contains one Xholsite in the PolgA^(D257Aneo) allele. We determined that 95% (40 out of42) of ES cell clones contained the desired double-base substitution asdetermined by digestion of PCR products with Xhol, followed by agarosegel electrophoresis.

We next expanded seven correctly targeted ES cell clones and injectedthese cells into blastocysts derived from C57BI/6J (B6) female mice.Injected blastocysts were implanted in pseudo-pregnant females forgeneration of chimeric mice. Eleven chimeric mice were identified asdetermined by coat color. Of these, five chimeras representing threedifferent ES cell clone lines resulted in germline transmission of thePolgA^(D257Aneo) allele when mated to B6 females. Mice carrying one copyof the PolgA^(D257Aneo) allele were healthy, fertile, and were used togenerate homozygote polgA^(D257Aneo/D257Aneo) mice.

We performed POLG western analysis in heart, skeletal muscle and celllines derived from polgA^(D257Aneo) mice and determined that POLGprotein is expressed in both heterozygous and homozygouspolgA^(D257Aneo) mice, suggesting that neither the neo cassetteintroduced at the PolgA gene, nor the PolgA^(D257A) doublebase-substitution impairs transcription or translation of the PolgAgene. Indeed, expression of the PolgA^(D257Aneo) allele was confirmed byallele specific RT-PCR (data not shown). Heterozygous PolgA^(D257Aneo)mice were also crossed to the mixed 129/ICR background Crerecombinase-expressing strain TgN(CMV-Cre)1AN (A. Nagy et al., Curr.Biol. 8:661-664, 1998). Cre is driven from a CMV promoter in thisstrain, which has been used previously to remove selectable markersflanked by loxP sites (A. Nagy et al., supra, 1998). Excision of the neocassette, as determined by PCR of genomic DNA, was observed in alltissues tested (brain, heart, kidney, liver, spleen, tail).Additionally, these animals transmitted the rearranged (neo excised)PolgA^(D257A) locus to their offspring. These animals differ fromwild-type animals at the PolgA locus by the specific two-base pairalteration introduced in positions 1054-1055 and by a small region (171bp) containing a loxP site left after the Cre-mediated recombination.Mixed background 129Sv/ICR PolgA^(D257Aneo/+) mice were crossed with B6mice through up to four backcross generations.

Generation and Analysis of Mouse Embryonic Fibroblasts (MEFs).

Females were mated and checked daily for mucous plugs. Animals weresacrificed at 12.5 dpc and embryos isolated from the uterus. Heads wereremoved for DNA isolation and genotyping. Embryo bodies were mincedusing sterile scissors and placed in 3 mL syringes with 1 mL of trypsin.The material was passed through an 18-inch gauge needle and incubatedfor 10 minutes at 37° C. Following trypsin inactivation, the embryonicmixture was transferred into a 6 cm dish. After 24-48 hours, cells weretransferred to a 10 cm dish. Cells were frozen upon confluence aspassage 1.

For studies of replicative senescence, cultures were passaged using a1:4 subculture regimen and incubated at 37° C. in a hypoxic atmosphereof 93% N₂, 5% CO₂, and 2% O₂ or normoxic atmosphere of 75% N₂, 5% CO₂,and 20% O₂. Cells were cultured in Ham's F10 nutrient mixture (LifeTechnologies) supplemented with 10% fetal bovine serum (Hyclone), 20 mML-glutamine (Gibco BRL), and penicillin/streptomycin (Gibco BRL).

Sequencing of mtDNA.

Mitochondria were isolated from heart, duodenum and liver fromindividual animals (2/group, 5-6 months of age) by differentialcentrifugation as previously described (Y. Hatefi, Methods Enzymol.53:48-54, 1978). For sperm analysis, sperm was isolated by incubatingsliced cauda epididymis in PBS at 37° C. for 30 minutes followed byremoval of the cauda and sperm pelleting by centrifugation. DNA wasisolated by overnight digestion with 0.5 mg/ml proteinase K in TENSbuffer (50 mM Tris, pH 8.0; 100 mM EDTA; 200 mM NaCI; 1 % SDS), followedby ethanol precipitation. DNA preparations were digested with DrallI andBglll followed by treatment with Exonuclease IlIl in order to degradeany contaminating nuclear DNA. A mitochondrial DNA fragment of 525 bpwas amplified by PCR (MTC1 primer, gCCAACTAgCCTCCATCTCATACTT, nt15196-15220 in B6 mtDNA, SEQ ID NO: 4; MTC2 primer,gggCgggTTgTTggTTTCAC, nt 15701-15720, SEQ ID NO: 5) using Easy-AHi-Fidelity™ cloning enzyme (Stratagene). PCR products were cloned intopCR4-TOPO™ vector using TOPO™ TA Cloning Kit (Invitrogen). One hundredninety-two colonies of each individual tissue source were grown andplasmid DNA isolated using the QIAPREP TURBO 96 Mini-prep Kit (Qiagen).Plasmid DNA was sequenced using standard M13 forward and reverseprimers. Reaction products were purified using Clean-seq magnetic beads(Agencourt) and sequenced on an ABI 3730xl capillary sequencer. DNAsequences were aligned to the B6 mtDNA reference sequence (Accession:NC_(—)005089) using Aligner software (CodonCode™) for identification ofmutations. Each tissue source yielded approximately 180,000 bases ofsequence, totaling over 300,000 bases per tissue per genotype for thetwo pair analysis.

Histological Procedures.

Tissues and organs were collected from mice following sacrificeaccording to a standard necropsy protocol. The entire thymus was fixedin 10% buffered formalin solution, as were both testes. A segment ofeach duodenum and small intestine, 3.5 cm long beginning at the pylorus,was opened longitudinally, pinned to a paper card, and fixed for atleast 24 hours. Before further processing, the intestine was rolled intoa spiral and pinned in position. Each testis was sliced longitudinallyinto two equal pieces following fixation. Fixed tissues were processedthrough a series of ethanol solutions of increasing concentrations,cleared in xylene, and embedded in paraffin. Prior to the final step ofembedment, the rolled intestines were sliced into two halves,representing a division of the intestine into two longitudinal pieces.The two halves were embedded together so that subsequent sections weremirror images. Sections of all tissues were cut at 5 Um and two sliceswere mounted on Fisher Superfrost Plus slides.

For histopathology of inner ear, mice were decapitated under deepanesthesia with 240 mg/kg tribromoethanol. After removing the temporalbones, the cochleae were perfused with 2% paraformaldehyde and 2.5%glutaraldehyde in phosphate buffered saline (PBS) through the round andoval windows, immersed in the same fixative for 24 hours, anddecalcified in 10% EDTA (pH 7.2) for 24 hours. The cochleae were thenrinsed with PBS, dehydrated through a graded series of alcohol, andembedded in epoxy resin. Thin sections cut parallel to the modiolus werestained with 0.5% toluidine blue and were observed under a lightmicroscope.

TUNEL Staining.

Paraffin was removed from the sections with xylene followed by ethanoland rinsed in water. TUNEL staining for apoptotic nuclei was performedusing the DeadEnd™ Colormetric TUNEL System (Promega, Madison) accordingto the manufacturer's instructions. Following preliminary studies,permeabilization with proteinase K was omitted as unnecessary forstaining in these tissues. Labeling reactions were performed for 60minutes at 25° C. in a humidified chamber. Color development wasaccomplished with DAB for 8 minutes. Duplicate sections werecounter-stained with hematoxylin and all slides were covered with acoverglass mounted with Permount.

Sections were examined with a Leica DM LB microscope using a 40×objective. Positively-stained apoptotic nuclei were counted in sectionsof intestine, thymus, and testes by direct observation. For sections ofintestine, the entire length represented in the sections were viewed andcounted. The number of villi represented in the length were counted.under the 10× objective. Apoptosis was evaluated as the number ofpositively-stained nuclei per 100 villi. Sections of thymus were sampledby random movement of the mechanical microscope stage to bring new,non-overlapping areas into view. Five fields of 300 μm×225 μm, as seenusing the 40× objective, were sampled for each thymus (total=0.36 mm²).Counts were standardized from the mean number of apoptotic-positivecells per field to number per 1×10⁵μm². Apoptotic nuclei in testes werecounted in a representative longitudinal cross-section of one testisfrom each mouse. The number of cross-sections of tubules for each samplewas determined from counts on low magnification mosaic photos of eachtestis. The number of apoptotic nuclei per cross-section of seminiferoustubule was calculated for each mouse.

Assessment of Hearing Function.

Hearing function was tested in 2 month old and 9 month old wild-type andD257A mice. Auditory brainstem responses (ABRs) were measured with atone burst stimulus (4, 8, and 16 kHz) using an ABR recording system(Intelligent Hearing System, Miami, Fla.). Animals were anesthetizedwith a mixture of xylazine hydrochloride (10 mg/kg, i.m.) and ketaminehydrochloride (40 mg/kg, i.m.), and needle electrodes were placedsubcutaneously at the vertex (active electrode), beneath the pinna ofthe measured ear (reference electrode), and beneath the opposite ear(ground). The stimulus duration, presentation rate, and rise/fall timewere 3 ms, 19.3/s, and 1 ms respectively. Responses of 1024 sweeps wereaveraged at each intensity level (5 dB steps) to assess threshold.Threshold was defined as the lowest intensity level at which a clearreproducible waveform was visible in the trace. Five mice per genotypeat each age were used for the study.

Measurement of Heart Function.

Transthoracic echocardiography was performed using an Acuson Sequoia(Siemens) ultrasonograph with a 15-MHz transducer. For acquisition oftwo-dimensional guided M-mode images at the tips of papillary musclesand Doppler studies, mice were sedated by IP administration of 100 mg/kgketamine and maintained on a heated platform in a left lateral decubitusposition. The chest was shaved and prewarmed coupling gel applied.Transmitral velocities were measured using Doppler pulse wave imaging.All images were saved to an on-board optical disk.

End diastolic and systolic left ventricular (LV) diameter as well asanterior and posterior wall (AW and PW respectively) thicknesses weremeasured on line from M-mode images using the leading edge-to-leadingedge convention. All parameters were measured over at least threeconsecutive cardiac cycles and averaged. Left ventricular fractionalshortening was calculated as [(LV diameter_(diastole) −LVdiameter_(systole))/LV diameter_(diastole)]×100 and LV mass wascalculated by using the formula [1.05×((PosteriorWall_(diastoLe)+Anterior Wall_(diastoLe)+LV diameter_(diastole))³−(LVdiameter_(diastole))³)]. Relative wall thickness was calculated as 2×Posterior wall_(diastole)/LV diameter_(diastole). Heart rate wasdetermined from at least three consecutive intervals from the pulse waveDoppler tracings of the LV outflow tract. Isovolumic relaxation time wasmeasured as the time from the closing of the aortic value to the openingof the mitral value from pulse wave Doppler tracings of the LV outflowtract and mitral inflow region. The same person obtained all images andmeasures.

Measurement of DNA and RNA Oxidation Markers.

Liver nuclei-pellets were dissolved in 3M guanidine isothiocyanate, 0.2%sodium N-lauroylsarcosinate, 20 mM Tris, pH 7.5 containing 10 mM freshlydissolved deferoxamine mesylate by pipetting on ice. Fat and proteinswere removed by extraction with phenol/chloroform/isoamylalcohol(25:24:1). The aqueous phase was withdrawn and afterchloroform/isoamylalcohol (24:1) extraction to remove traces of phenol,nucleic acids were precipitated by addition of isopropanol (−80° C., 1h). After washing in 70% ethanol, nucleic acids pellets were dissolvedin water containing 30 pM deferoxamine mesylate and hydrolysed usingnuclease P₁ and alkaline phosphatase. Nucleoside content of guanosine(RNA), 8-hydroxyguanosine (RNA), 2′-deoxyguanosine (DNA) and8-hydroxy-2′-deoxyguanosine (DNA) was determined using HPLC-EC-UV (T.Hofer and L. Moller, Chem. Res. Toxicol. 15:426-432, 2002).

Mitochondrial and Cytosolic Extracts.

Animals were sacrificed at 3 months and 9 months of age. Animals wereanesthetized with isoflurane gas, which was administered via inhalationusing a precision vaporizer at 3%. Following anesthesia the chest cavitywas opened and the heart was removed rapidly, followed by liver, thymus,testes, duodenum, skeletal muscle, and inner ear tissue. Mitochondrialand cytosolic protein fractions were isolated using differentialcentrifugation. Briefly, heart and liver were finely minced andhomogenized on ice in 1:10 (wt/vol) ice-cold isolation buffer (heartbuffer composition: 220 mM mannitol, 70 mM sucrose, 1 mM EDTA, and 10 mMTris-HCl, 0.2% fatty acid free BSA, pH 7.4; liver buffer composition:210 mM mannitol, 70 mM sucrose, 1 mM EDTA, 5 mM HEPES, pH 7.35) using aPOTTER-ELVEHJEM glass homogenizer. The homogenates from heart and liverwere centrifuged for 10 minutes at 700 g and 1000 g, respectively, andthe resulting supernatants were centrifuged for 20 minutes at 8,000 g(heart) or 10,000 g (liver). The 8,000 g and 10,000 g supernatants(representing crude cytosolic fractions) were frozen at −80° C foranalysis. Pellets were resuspended in a small volume of isolation bufferand centrifuged at 8,000 g (heart) or 10,000 g (liver) for 10 minutes.All centrifugation steps were carried out at 4° C. The finalmitochondrial pellets were suspended in isolation buffer (without BSA,for heart mitochondria and without EDTA, for liver mitochondria) andused immediately for measurement of mitochondrial H₂O₂ and ATPproduction. For cytosolic extraction from thymus, duodenum, testes andgastrocnemius muscle, tissues were homogenized in 1:10 (wt/vol) (1:5 forskeletal muscle) ice-cold isolation buffer (20 mM HEPES, 10 mM KCI, 250mM sucrose, 1.5 mM MgCl₂, 1 mM EDTA, pH 7.4). The homogenates werecentrifuged at 800-1000 g depending on the tissue for 10 minutes, andthe resulting supernatants were centrifuged at 10,000 g for 20 minutes.The supernatants (crude cytosol) were immediately frozen at −80° C. forfurther biochemical analysis.

Determination of Cytosolic Mono- and Oligonucleosomes.

Endogenous endonucleases activated during apoptosis cleavedouble-stranded DNA in the linker region between nucleosomes to generatemono- and oligonucleosomes of 180 bp or multiples. Apoptotic DNAfragmentation was quantified in the thymus and testes by measuring theamount of cytosolic mono- and oligonucleosomes using a Cell Death™ ELISAkit (ROCHE MOLECULAR BIOCHEMICALS, Germany) with the peroxidasesubstrate ABTS (A. Dirks and C. Leeuwenburgh, Am. J. Physiol. Regul.lntegr. Comp. Physiol. 282:R519-R527, 2002; S. K. Lee, et al.,Anticancer Res. 22:97-102, 2002). All samples were run in triplicate andthe means expressed as arbitrary OD units normalized to milligram ofcytosolic protein, with sample protein concentrations determined by theBradford method (M. M. Bradford, Analyt. Biochem. 72:248-254, 1976).

Determination of Cleaved Caspase-3 Content.

The active form of caspase-3, cleaved caspase-3, was quantified byWestern blotting. Activation of caspase-3 requires proteolyticprocessing of its inactive zymogen into activated fragments. Thespecific antibody used (see below) detects endogenous levels of thelarge fragment (17/19 kDa) of activated caspase-3 resulting fromcleavage adjacent to Asp175. For quantification of cleaved caspase-3 byWestern blot analysis, cytosolic proteins from the thymus, testes,duodenum and gastrocnemius muscle were separated using 4-20% PAGEr® Goldpre-cast Tris-glycine gels (BioWittaker Molecular Applications,Rockland, Me., USA) under denaturing conditions, and then transferred tonitrocellulose membranes (0.2 μm, Trans-Blot® Transfer Medium, Bio-RadLaboratories, Calif., USA). Protein concentration was determined usingthe Bradford assay (M. M. Bradford, supra, 1976), and subsequentlynormalized so that the protein content among samples was identical.Subsequently, 20 μl of sample was loaded to each well. A 15 μl sample ofHL-60 cell extract induced with etoposide (EMD Biosciences, Inc., SanDiego, Calif.) was used as a positive control. Only protein bands withinan individual gel were used for comparisons. Membranes were blocked for1 h using a blocking solution containing PBS and 5% milk. Membranes werethen incubated overnight in the 5% blocking solution containing therabbit monoclonal primary anti-cleaved caspase-3 antibody (CellSignaling, Beverly, Mass., USA) with an appropriate dilution (1:100 forgastrocnemius and testes, 1:300 for thymus, and 1:1000 for duodenum).The following day membranes were incubated for 1 hour at roomtemperature with anti-rabbit IgG horseradish peroxidase-linked wholesecondary antibody (1:1000, Amersham Biosciences UK Ltd, Amersham, UK).Specific protein bands were visualized using ECL reagent (AmershamPharmacia Biotech, UK). The resulting Western blots were exposed to film(Hyperfilm™ ECI™, Amersham Pharmacia Biotech, UK) and analyzed using theKODAK Imaging System (Kodak 440CF). Values were expressed as arbitraryOD units. Triplicate measurements were taken and the resulting means(±SEM) were used for analysis.

Oxidant Production.

H₂O₂ production was measured in intact heart and liver mitochondria overa period of 15 minutes at 37° C. following the method of Barja (G.Barja, J. Bioenerq. Biomembr. 34:227-233, 2002). Briefly, incubationbuffer (145 mM KCI, 30 mM HEPES, 5 mM KH2PO4, 3 mM MgCl₂, 0.1 mM EGTA,and 0.1% fatty-acid free BSA, pH 7.4) was added to test tubes followedby the addition of mitochondria (0.25 mg protein/ml), horseradishperoxidase (5.7 U/ml), homovanilic acid (0.1 mM), and substrate (2.5 mMpyruvate/ malate) so that the total volume was equal to 1.5 ml. Thetubes were incubated in a shaking water bath at 37° C. for 15 minutes,and the reaction was stopped by placing the tubes in ice and adding 0.5ml cold stop solution (0.1 M glycine, 25 mM EDTA-NaOH, pH 12.0).Fluorescence was determined at 312 nm excitation and 420 nm Emissionusing a SPECTRAmax Gemini™ XS dual-scanning microplatespectrofluorometer (Molecular Devices, Sunnyvale, Calif.). Arbitraryfluorescence units were converted to known amounts of H₂O₂ using aglucose-glucose oxidase standard curve. All measurements were performedin duplicate, and results were expressed as nmol of H₂0₂ /minl mgprotein. ATP production.

Mitochondria isolated from heart and liver were used immediately afterisolation to determine mitochondrial ATP content and rate of ATPproduction following the method of Drew (B. Drew and C. Leeuwenburgh,Am. J. Physiol. Regul. Integr. Comp. PhVsiol. 285:R1259-R1267, 2003).ATP production was determined using a luminometer (model TD-20/20,Turner Designs, Sunnyvale, Calif.), employing an assay that utilizesfirefly luciferase, which fluoresces in proportion to the presence ofATP and d-luciferin. In order to determine ATP content, freshly isolatedmitochondria were added to a cuvette containing 1 mM pyruvate, 1 mMmalate, and a luciferin-luciferase ATP monitoring reagent (ATPDetermination Kit A-22066, Molecular Probes, Eugene, OR). This wasfollowed immediately by the addition of 2.5 mM ADP to determine ATPproduction. A blank cuvette containing no sample was assayed to accountfor nonspecific ATP production, and known concentrations of ATP wereused to establish a standard curve. All mitochondrial samples wereassayed in triplicate, and an average of these results was used inquantifying ATP content and rate of production. Results (mean±SEM) forATP content were expressed as nmol ATP/mg protein, and for ATPproduction as nmol ATP/mg protein/min.

Statistical Analysis.

All analyses were performed in triplicate and the means obtained wereused for independent t-tests. Statistical analyses were carried outusing the Prism 4.0 statistical analysis program (GraphPad, San DiegoCalif.). Statistical significance was set at P<0.05. All data arereported as mean±SEM.

Those of ordinary skill in the art will readily appreciate that theforegoing represents merely certain preferred embodiments of theinvention. Various changes and modifications to the procedures andcompositions described above can be made without departing from thespirit or scope of the present invention, as set forth in the followingclaims below.

1. A transgenic mouse model for mammalian aging comprising a mousehaving a genomic mutation in the exonuclease domain of a mitochondrialDNA polymerase gamma (Polg) gene, wherein the mutation results inelevated mitochondrial mutation frequency in at least two tissues. 2.The mouse model of claim 2 wherein the mutation is selected from thegroup consisting of a single or double base pair nucleotidesubstitution.
 3. The mouse model of claim 1 having a genomic mutation inthe exonuclease domain II (ExoII) of a mitochondrial DNA polymerasegamma (Polg) gene, wherein the mutation is a nucleotide change withinnucleotide residues 1026 through 1067 of the mouse Polg gene.
 4. Themouse model of claim 1 wherein the impairing of the exonuclease domainactivity results in the expression of a DNA proof-reading deficientversion of the mitochondrial DNA polymerase gamma (Polg) gene andaccumulation of mitochondrial DNA mutations in both mitotic andpost-mitotic mouse tissues, which is correlated with the activation ofcaspase-3 and the induction of apoptosis in mouse tissues.
 5. The mousemodel of claim 3 wherein a double base substitution results in a changein the coding of amino acid residue 257 from an aspartic acid (D) to analanine (A) in the exonuclease domain II (ExoII) of Polg.
 6. The mousemodel of claim 1 wherein the mouse is D257A.
 7. The mouse model of claim1 wherein the mouse Polg gene sequence has an Accession No: NM_(—)017462as set forth in SEQ ID NO:
 1. 8. The mouse model of claim 1 wherein themutated mouse Polg gene sequence has the mutation as set forth in SEQ IDNO:
 3. 9. The mouse model of claim 1 wherein the mouse exhibits symptomsof accelerated or premature aging compared to a mouse not having themutation in the exonuclease domain of the Polg gene.
 10. The mouse modelof claim 1 wherein the aging symptoms are selected from the groupconsisting of abnormalities in tissues of high cellular turnover, heartdysfunction, graying hair and alopecia, auditory function loss, cochleardegeneration, immune cell loss, anemia, male germ cell loss leading tolack of sperm and infertility, skeletal muscle mass loss (sarcopenia),neurodegeneration, increased presence of apoptotic markers, and loss ofbone mass.
 11. A method of screening for a potentially therapeutic agentuseful for delaying the onset of aging-related symptoms, the methodcomprising the steps of: (a) providing a mouse model of claim 1, whereinthe mouse exhibits aging-related symptoms; (b) administering the agentto the mouse model; and (c) determining whether the agent is capable ofdelaying the onset of aging-related symptoms in the mouse model treatedwith the agent compared to an untreated mouse model.
 12. The method ofclaim 11 wherein the aging-related symptoms are selected from the groupconsisting of abnormalities in tissues of high cellular turnover, heartdysfunction, graying hair and alopecia, auditory function loss, cochleardegeneration, immune cell loss, anemia, male germ cell loss leading tolack of sperm and infertility, skeletal muscle mass loss (sarcopenia),bone loss, neurodegeneration and increased presence of apoptotic stressmarkers.
 13. The method of claim 11 wherein the age-related symptoms areselected from the group consisting of altered hearing function, alteredheart function, loss of bone, loss of muscle mass and induction ofapoptosis.
 14. The method of claim 11 wherein the therapeutic agent is agenetically-, a pharmaceutical- or a dietary-based agent.
 15. A methodof screening for a potentially therapeutic agent useful for treatingmedical conditions comprising progressive external ophthalmoplegia,sensorimotor polyneuropathy, ataxia, Parkinson's syndrome or earlymenopause defined by mitochondrial DNA mutations in a POLG gene, themethod comprising the steps of: (a) providing a mouse model of claim 1,wherein the mouse exhibits symptoms of progressive externalophthalmoplegia sensorimotor polyneuropathy, ataxia, Parkinson'ssyndrome or early menopause; (b) administering the agent to the mousemodel; and (c) determining whether the agent is capable of improvingsymptoms for any of the medical conditions of step (a) in the mousemodel treated with the agent compared to an untreated mouse model.
 16. Amethod for generating a mouse model for mammalian aging, comprising thesteps: (a) introducing into a mouse a germlne transmission of a mutationan exonuclease domain of a mitochondrial DNA polymerase gamma (POlG)gene, wherein the mutation results in a decrease in the POlG exonucleaseactivity, and wherein the mutation results in the production of aPolgA^(exo−/+) mouse; (b) crossbreeding the PolgA^(exo−/+) mouse of step(a); and (c) generating a homozygous PolgA^(exo−/+) mouse which exhibitsat least one symptom of aging, wherein the symptoms of aging areselected from the group consisting of abnormalities in tissues of highcellular turnover, heart dysfunction, graying hair and alopecia,auditory function loss, cochlear degeneration, immune cell loss, anemia,male germ cell loss leading to lack of sperm and infertility, skeletalmuscle mass loss (sarcopenia), loss of bone mass, neurodegeneration andincreased presence of apoptotic stress markers.
 17. The method of claim16 wherein the mutation is a double base substitution (AC to CT) atnucleotide residues 1054 to 1055 of the mouse Polg gene.